Compositions and methods for detecting allogeneic matter in a subject

ABSTRACT

The present disclosure provides a panel of nucleic acid molecule primers specific for HLA-specific alleles and other genetic polymorphisms, which are useful for quantitatively amplifying these markers to detect, diagnose, and monitor individuals who have or are at risk of certain disease conditions, such as autoimmune disease, proliferative disease, infectious disease, allograft rejection, or pregnancy-related pathologies.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under AI041721, AI045659, AR048084, AI045952 and HL117737 awarded by the National Institutes of Health. The government has certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 411D1_SEQUENCE_LISTING.txt. The text file is 16.3 KB, was created on Feb. 19, 2020, and is being submitted electronically via EFS-Web.

BACKGROUND 1. Technical Field

The present disclosure relates to compositions and methods for quantitatively amplifying nucleic acid molecules from biological samples and, more particularly, to a panel of nucleic acid molecules useful for quantitative amplification of target markers for detecting, diagnosing, or monitoring individuals who have or are at risk of certain disease conditions, such as autoimmune disease, proliferative disease, infectious disease, allograft rejection, or pregnancy-related pathologies.

2. Description of Related Art

Individuals harbor small amounts of foreign cells or DNA, referred to as microchimerism (“Mc”). Acquisition of Mc occurs naturally during pregnancy through transplacental cell trafficking between mother and fetus, although Mc may be acquired through other sources (e.g., blood transfusion, organ transplantation). An adult woman may have acquired Mc from her own mother (maternal Mc, “MMc”) while she herself was a fetus. This “graft,” acquired during fetal immune system development, can remain in her system into adulthood (Maloney et al., J. Clin. Invest. 104:41, 1999; Lambert et al., Arth. Rheu. 50:906, 2004) and represents a pre-existing inhabitant as she experiences pregnancy herself. During subsequent pregnancies, new fetal sources of microchimerism (fetal Mc, “FMc”) can be acquired and also remain for years. The interactions of each of these grafts with the host, and with other pre-existing inhabitants, may be beneficial or detrimental to an individual.

Disease and Mc may have a functional connection since persistence of Mc has been shown to be associated both positively and negatively with certain disease states, such as autoimmune disease (Evans et al., Blood 93:2033, 1999; Yan et al., Arth. Rheu. 63:640, 2011), malignancy (Gadi, Breast Cancer Res. Treat. 121:241, 2010; Gadi et al., PLoS ONE 3:e1706, 2008), and transplant rejection (Gadi et al., Clin. Chem. 52:379, 2006; Gadi et al., Transpl. 92:607, 2011). In autoimmunity, higher detection rates and concentrations of Mc suggest a possible allo-autoimmune or auto-alloimmune functionality (Gammill and Nelson, Int. J. Dev. Biol. 54:531, 2010). In the case of malignancy, lower detection rates and concentrations of Mc in cancer cases suggest a possible graft-versus-tumor effect (Gadi, Cancer Lett. 276:8, 2009). In transplantation, the presence of Mc may serve as a biomarker for monitoring allograft survival (Gadi et al., 2006, 2011).

From the foregoing, a need is apparent for improved compositions and methods for sensitively and quantitatively identifying allogeneic cells, tissues, and nucleic acids resulting from medical conditions or interventions.

BRIEF SUMMARY

In one aspect, the present disclosure provides compositions comprising a forward nucleic acid molecule, a reverse nucleic acid molecule, and a probe nucleic acid molecule comprising a fluorophore and a quencher, wherein each individual composition is specific for HLA-B*44, HLA-DRB1*01, HLA-DRB1*15/16, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07, HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*10, HLA-DRB1*14, HLA-DRB4, HLA-DQA1*01, HLA-DQA1*03, HLA-DQA1*05, HLA-DQB1*02, HLA-DQB 1*03, HLA-DQB1*04, HLA-DQB1*06, SE-HR, SE-LR, GSTT1, AT3, or Tg, as set forth in Table 1, and wherein each nucleic acid molecule has a length ranging from about 10 nucleotides to about 35 nucleotides.

In another aspect, the present disclosure provides methods for quantitating microchimerism, monitoring allograft rejection, anti-malignancy effects, engraftment dominance, or the like, wherein the methods involve amplifying nucleic acid molecules of a target biological sample using one or more specific nucleic acid compositions listed in Table 1 and analyzing for the presence or absence of certain specific amplified markers in the target biological sample to monitor or assess microchimerism, monitoring allograft rejection, anti-malignancy effects, engraftment dominance, or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of the longitudinal assessment of donor DNA concentrations and acute transplant rejection (ACR). High concentrations of donor DNA (given in qEq/106 host genomes), as measured with the DQB1*06 nucleic acid molecule composition in Q-PCR, were detected in serum from a patient at points correlating with ACR in pancreas and kidney specimens (*) or pancreas alone (**). Day 196 was negative for ACR in the pancreas.

FIG. 2 shows a bar graph illustrating quantitative expression of maternal microchimerism in different tissues, peripheral blood mononuclear cells (PBMCs), and bone marrow (B. marrow) from a proband with systemic sclerosis. Tp=autologous hematopoietic stem cell transplantation.

DETAILED DESCRIPTION

The present disclosure provides compositions of nucleic acid molecules for use in quantitatively detecting the presence of allogeneic material, such as cells or DNA, in a subject. Such compositions and methods can be useful in detecting microchimerism, transplant rejection, malignancy relapse, potential pathology associated with pregnancy, infectious disease, or the like. Exemplary compositions include a forward nucleic acid molecule, a reverse nucleic acid molecule, and a probe nucleic acid molecule comprising a fluorophore and a quencher, wherein each composition is specific for a particular HLA allele, for particular polymorphisms of an HLA allele, or for other genetic polymorphisms with different alleles or a null allele.

By way of background, some cells may (and often times do) traffic between a mother and fetus during pregnancy. Surprisingly, small numbers of these allogeneic cells can persist in their respective hosts decades later. Microchimerism (Mc) refers to an individual harboring a small number of cells, or DNA, derived from another individual. As noted above, the instant disclosure provides compositions and methods for examining a wide breadth of consequences of naturally-acquired Mc across all of human health (Adams and Nelson, JAMA 291:1127, 2004).

For example, Mc may have an effect on or have a role in autoimmune disease. In a first study of Mc, elevated levels of fetal Mc in blood were found in women with scleroderma compared to healthy women (Nelson et al., Lancet 351:559, 1998). Fetal Mc (FMc) has since been investigated in primary biliary cirrhosis, thyroiditis, Sjögren's syndrome, polymorphic eruption of pregnancy and rheumatoid arthritis. Maternal Mc (MMc) can be found in her adult progeny (Maloney et al., J. Clin. Invest. 104:41, 1999). MMc has been studied in neonatal lupus, systemic lupus and myositis. In neonatal lupus with heart block, maternal cells in the heart were cardiac myocytes (Stevens et al., Lancet 362:1617, 2003). Thus, microchimeric cells could be targets or alternatively could help repair damaged tissues. FMc may be beneficial during pregnancy in women with rheumatoid arthritis as elevated levels, assessed by Q-PCR, significantly correlated with pregnancy-induced amelioration of arthritis (Yan et al., Arthritis Rheum. 54:2069, 2006).

In cancer, hematopoietic cell transplantation (HCT) donor cells provide an advantage against recurrent leukemia and other malignancies. By analogy, FMc might be responsible for protection from breast cancer observed in women who have had prior pregnancies compared to those who have not. In other types of malignancies, anecdotal reports indicate that microchimeric cells may cause or result in malignancy.

For infectious disease, T lymphocytes are an important determinant of immune reactions between one's own cells and foreign cells. Human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) are characterized by critical deficiencies in CD4+ T cells. MMc can be examined in HIV and AIDS by employing HLA and other genetic polymorphism specific Q-PCR to quantify MMc in men with HIV to correlate results progression as compared to non-progression to AIDS.

In the field of transplantation, iatrogenic chimerism can result. But, donor Mc may facilitate graft acceptance. Until recently, donor Mc was measured as male DNA in female recipients, but it is now clear that women commonly have male DNA from prior pregnancies. Thus, the application of specific Q-PCR assays of this disclosure provides a major step forward for these studies. In hematopoietic cell transplantation (HCT), graft-versus-host disease (GVHD) occurs more often if the donor is a woman with prior pregnancies. Female apheresis products were found to contain male Mc, consistent with the idea that fetal Mc contributes to GVHD (Adams et al., Blood 15:3845, 2003). In kidney, pancreas and islet transplantation embodiment, the panel of Q-PCR assays of the instant disclosure are useful for testing serial serum samples, which provides a non-invasive test for early rejection (see Example 1). The Q-PCR assays of the instant disclosure may also be used to monitor the fate of co-transplanted hematopoietic cells as it affects a kidney allograft from the same donor.

In the context of pregnancy, the compositions and methods of the instant disclosure are useful for identifying changes in the maternal carriage of fetal cells throughout the course of normal pregnancy (see Example 2). In certain embodiments, the tools and methods of the instant disclosure can be used to examine pathologic conditions associated with pregnancy (pre-eclampsia, early fetal loss, and abnormally genetic fetuses, as examples) and their association with deviations from normal pregnancy patterns.

Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.

In the present description, the terms “about” and “consisting essentially of” mean±20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms “include,” “have” and “comprise” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

As used herein, “fluorophore” refers to a molecule that emits light of a certain wavelength after having first absorbed light of a specific, but shorter, wavelength, wherein the emission wavelength is always higher than the absorption wavelength.

As used herein, “quencher” refers to a molecule that accepts energy from a fluorophore in the form of light at a particular wavelength and dissipates this energy either in the form of heat (e.g., proximal quenching) or light of a higher wavelength than emitted from the fluorophore (e.g., FRET quenching). Quenchers generally have a quenching capacity throughout their absorption spectrum, but they perform best close to their absorption maximum. For example, Deep Dark Quencher II absorbs over a large range of the visible spectrum and, consequently, efficiently quenches most of the commonly used fluorophores, especially those emitting at higher wavelengths (like the Cy® dyes). Similarly, the Black Hole Quencher family covers a large range of wavelengths (over the entire visible spectrum and into the near-IR). In contrast, Deep Dark Quencher I and Eclipse® Dark Quencher effectively quench the lower wavelength dyes, such as FAM, but do not quench very effectively those dyes that emit at high wavelengths.

As used herein, “target nucleic acid molecules” and variants thereof refer to a plurality of nucleic acid molecules that may be found in certain biological samples but may be missing from others, depending on the genetic make-up of the subject and the extent of microchimerism present. Nucleic acid molecules include those from natural samples (e.g., a genome, RNA), or the target nucleic acid molecules may be synthetic samples (e.g., cDNA), recombinant samples, or a combination thereof.

As used herein, a “nucleic acid molecule primer” or “primer” and variants thereof refers to short nucleic acid sequences that a DNA polymerase can use to begin synthesizing a complementary DNA strand of the molecule bound by the primer. A primer sequence can vary in length from 5 nucleotides to about 50 nucleotides in length, from about 10 nucleotides to about 35 nucleotides, and preferably are about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. In certain embodiments, a nucleic acid molecule primer that is complementary to a target nucleic acid of interest can be used to initiate an amplification reaction, a sequencing reaction, or both.

For example, for quantitative PCR, the combination of an upstream or forward primer (5′) and a downstream or reverse primer (3′) complementary to a target sequence of interest (e.g., HLA-B, HLA-DRB) can be used to prime an amplification reaction to obtain the sequence of the nucleic acid molecule of interest. A “probe” oligonucleotide sequence is similar to a primer except that it will further contain a fluorophore molecule and a quencher molecule and hybridize to a target nucleic acid molecule of interest somewhere between the forward and reverse primer binding sites.

In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of this disclosure. However, upon reviewing this disclosure one skilled in the art will understand that the invention may be practiced without many of these details. In other instances, newly emerging amplification technologies, as well as well-known or widely available specific probe amplification methods and tools (e.g., Taqman® probes, Locked Nucleic Acid (LNA) probes, Molecular Beacon probes, Scorpions® primers, Hybridization probes, MGB probes, QuantiProbes, Resonsense probes, Light-up probes, HyBeacon® probes, LUX primers, Yin-yang probes, Amplifluor®), have not all been described in detail to avoid unnecessarily obscuring the descriptions of the embodiments of the present disclosure.

Various embodiments of the present disclosure are described for purposes of illustration, in the context of use with HLA-specific alleles and certain other genomic polymorphisms. But, as those skilled in the art will appreciate upon reviewing this disclosure, use with other target nucleic acid molecules may also be suitable.

In certain embodiments, the present disclosure provides methods for detecting, diagnosing, or monitoring the presence of allogeneic cells, tissues, or nucleic acid molecules that may be involved in or associated with a particular medical condition, such as autoimmune disease, neoplastic disorders, infectious disease, transplant rejection, and pathologies associated with pregnancy. In further embodiments, the methods of the instant disclosure are sensitive enough to detect one chimeric genome in 10⁵ host genomes or 10⁶ host genomes when amplifying specific target nucleic acid molecules in presence of many different nucleic acid molecules.

In further embodiments, the compositions and methods of this instant disclosure will be useful in detecting rare nucleic acid molecules or cells against a large background signal, such as when monitoring for Mc in autoimmune disease, infectious disease, malignancies, transplant subjects, pregnancy, or forensics. Additional embodiments may be used to quantify target nucleic acid molecules that may be indicative of response to therapy or may be useful in monitoring disease progression or recurrence. In yet other embodiments, these compositions and methods may be useful in detecting or monitoring target nucleic acid molecules after or during chemotherapy, autoimmune therapy, infectious disease therapy, or treatments of complications to pregnancy.

Representative nucleic acid molecule compositions of the present disclosure may be specific for HLA alleles, such as HLA-B*44, HLA-DRB1*01, HLA-DRB1*15/16, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07, HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*10, HLA-DRB1*14, HLA-DRB4, HLA-DQA1*01, HLA-DQA1*03, HLA-DQA1*05, HLA-DQB1*02, HLA-DQB1*03, HLA-DQB1*04, or HLA-DQB1*06 (see Table 1). Alternatively, nucleic acid molecule compositions of the present disclosure may be specific for other genetic polymorphisms, such as SE-HR, SE-LR, GSTT1, AT3, or Tg (see Table 1). In certain embodiments, each nucleic acid molecule has a length ranging from about 10 nucleotides to about 35 nucleotides.

Selecting nucleic acid molecule compositions of the present disclosure is not routine because many of the sequences tested did not work due to cross-reactivity with other genes. For example, the DRB1*11 specific primers and probes described in Example 1 did not work because multiple cross-reactivity did not allow for analysis of this marker. Alternative sequences for this marker also did not work. Similarly, DRB1*05 and DQB1*05 did not work, and a particular primer pair for DRB1*10 (see HLA-DRB1*10-2 SEQ ID NOS.:28 and 29 of Table 1) also did not work, as well as various other primers and probes (not shown) for the targets listed in Table 1.

The probe nucleic acid molecules of the compositions of the present disclosure are preferably dual labeled oligonucleotides that include a fluorophore (e.g., FAM, TET, HEX, Cy®3, Cy®3.5, Cy®5, Cy®5.5, TAMRA, Yakima Yellow®, ROX) and a quencher (e.g., Deep Dark Quencher I, Deep Dark Quencher II, DabCYL, Eclipse® Dark Quencher, Black Hole Quencher (BHQ-0, BHQ-1, BHQ-2, BHQ-3, TAMRA). For example, the instant disclosure provides HLA and other genetic polymorphism-specific quantitative PCR (Q-PCR) primers and probes for use in diagnostics, detection of medical conditions, or monitoring medical conditions.

An exemplary panel of primers and probes useful in the compositions and methods of the instant disclosure are provided in Table 1.

TABLE 1 qPCR Primers and Probes* Target Forward (5′-3′) Reverse (3′-5′) Probe (5′-3′) HLA-B*44 CCG CGG GTA TGA TCC AGG TAT CGG CTC AGA TCA CCA GGA (SEQ ID CTG CGG AGC G CCC AGC GCA A NO.: 1) (SEQ ID NO.: 2) (SEQ ID NO.: 3) HLA-DRB1*01 CAC GTT TCT TGT GCT GTC GAA TC CTC TTG GTT GGC AGC TTA AGT GCG CAC GG (SEQ ATA GAT GCA TCT T (SEQ ID NO.: 4) ID NO.: 5) TTC CAG CAA CC (SEQ ID NO.: 6) HLA-DRB1*15/16 CGT TTC CTG TGG GCA CGG ACT CGT CCC ATT GAA CAG CCT AA (SEQ CCT CCT GGT GAA ATG ACA CTC ID NO.: 7) TAT (SEQ ID CCT C (SEQ ID NO.: 8) NO.: 9) HLA-DRB1*03 CCA CGT TTC TTG T GCA GTA GTT TT CTC CTC CTG GAG TAC TCT ACG GTC CAC CCG  A C GTT ATG GAA GTA TC (SEQ ID NO.: 10) (SEQ ID NO.: 11) TCT GTC CAG GT (SEQ ID NO.: 12) HLA-DRB1*04 CGT TTC TTG GAG CG CAC GTA CTC CAC CCG CTC CGT CAG GTT AAA CA CTC TTG GTG CCC GTT GAA (SEQ (SEQ ID NO.: 13) (SEQ ID NO.: 14) ID NO.: 15) HLA-DRB1*07 CGT TTC CTG TGG C CCC GTA GTT AAG TGT CAT TTC CAG GGT AAG TA GTG TCT GCA TTC AAC GGG ACG (SEQ ID NO.:16) CAC (SEQ ID GAG C (SEQ ID NO.: 17) NO.: 18) HLA-DRB1*08 A CGT TTC TTG G TCT GCA GTA TAT AAC CAA GAG GAG TAC TCT ACG GGT GTC CAC GAG TAC GTG CGC GG (SEQ ID NO.: 19) CAG (SEQ ID TTC GAC AG (SEQ NO.: 20) ID NO.: 21) HLA-DRB1*09 G CAC GTT TCT C CCC GTA GTT T TCT CCT CTT TGA AGC AGG A TG TCT GCA GGT TAT AGA TGC (SEQ ID NO.: 22) CAC (SEQ ID CTC TGT GCA GAT NO.: 23) (SEQ ID NO.: 24) HLA-DRB1*10-1 GGT TGC TGG AAA GTG TCC ACC AGT ACG CGC GCT GAC GCG (SEQ ID GCG GCA (SEQ ID ACG ACA GCG AC NO.: 25) NO.: 26) (SEQ ID NO.: 27) HLA-DRB1*10-2^(¶) CGG TTG CTG GAA GGT GTC CAC AGT ACG CGC GCT AGA  A GC G (SEQ CGC GG A  A (SEQ ACG ACA GCG AC ID NO.: 28) ID NO.: 29) (SEQ ID NO.: 27) HLA-DRB1*14 CGG CCT GCT GCG AAC CCC GTA CCG CCT CCG CTC GA A  C (SEQ ID GTT GTG TCT CAG GAG GT (SEQ NO.: 31) GCA A (SEQ ID ID NO.: 33) NO.: 32) HLA-DRB4*01 CAG GCT AAG TGT CCT GGT ACT TA TCT GAT CAG GAG TGT CAT TTC CCC CCA GGT CA GTT CCA CAC TCG C (SEQ ID NO.: 34) (SEQ ID NO.: 35) CTC CGT (SEQ ID NO.: 36) HLA-DQA1*01 C CTG GAG AGG AGC CAT GTT ACC TCC AAA TTT AAG GAG ACT GC TCT CAG TGC GCT GAA CTC AGO (SEQ ID NO.: 37) ACC (SEQ ID CCA C (SEQ ID NO.: 38) NO.: 39) HLA-DQA1*03 AA TTT GAT GGA GC AAA TTG CGG A TCT GCG GAA GAC GAG GAG TTC GTC AAA TCT CAG AGOCAA CTG TAT (SEQ ID (SEQ ID NO.: 41) CCA (SEQ ID NO.: 40) NO.: 42) HLA-DQA1*05 TTG CAC TGA CAA TGG TAG CAG AAC TTG AAC AGT ACA TCG CT A  TC CGG TAG AGT CTG ATT AA (SEQ (SEQ ID NO.: 43) TGG (SEQ ID ID NO.: 45) NO.: 44) HLA-DQB1*02 C GTG CGT CTT GTA CTC GGC AG CGT CAC CGC GTG AGC AGA AG GGC AGG CA CCG GAA CTC C (SEQ ID NO.: 46) (SEQ ID NO.: 47) (SEQ ID NO.: 48) HLA-DQB1*03 CGG AGC GCG TGC CGT GCG GAG AG GAC TTC CTT GTT A (SEQ ID CTC CAA CTG CTG GCT GTT CCA NO.: 49) (SEQ ID NO.: 50) GTA CTC G (SEQ ID NO.: 51) HLA-DQB1*04 TGC TAC TTC ACC CTA TTC CAG TCG GTT ATA GAT AAC GGG A A C TAC TCG GCG GTA TCT GGT CAC (SEQ ID NO.: 52) TCA A (SEQ ID ACC CCG (SEQ ID NO.: 53) NO.: 54) HLA-DQB1*06 GAC GTG GGG GTG CTG CAA GAT TTC CTT CTG GCT TAC CGC (SEQ ID CCC GCG GA (SEQ GTT CCA GTA CTC NO.: 55) ID NO.: 56) GGC AT (SEQ ID NO.: 57) SE-HR (QRRAA)† CCA GAA GGA CCT GTG TCT GCA CGG CCC GCC TCT CCT GGA GC (SEQ GTA GGT GT C (SEQ ID NO.: 60) ID NO.: 58) CAC  A G (SEQ ID NO.: 59) SE-HR (QKRAA)† CCA GAA GGA CCT GTG TCT GCA CGG CCC GCT TCT CCT GGA GC (SEQ GTA GGT GT (SEQ ID NO.: 63) ID NO.: 61) CAC  A G (SEQ ID NO.: 62) SE-LR (DERAA) CCA GAA GGA CAT GTG TCT GCA CGG CCC GCT CGT CCT GGA AG (SEQ GTA GGT GTC (SEQ ID NO.: 66) ID NO.: 64) CAC  A G (SEQ ID NO.: 65) GSTT1 TTC CAG GAG GCC GGG CAT CAG AAG GCC AAG CAT GAG (SEQ ID CTT CTG CTT TAT GAC TTC CCA CCT NO.: 70) G (SEQ ID NO.: 71) GCA (SEQ ID NO.: 72) AT3-S CCT CTC TCC ATA GCT TTA TAG CTT GGT TCA TAC AAG AAA ACT ATG AAA AGG AAA CCA CCC (SEQ ID AGA GA (SEQ ID AGG AGA GTA TG NO.: 75) NO.: 73) (SEQ ID NO.: 74) AT3-L CCT CTC TCC ATA GGA TTT TTT GTT CCC TCT ACC TGT AAG AAA ACT ATG TCT CGT TAA AAT TC (SEQ ID AGA GA (SEQ ID CTA AAT CAG NO.: 78) NO.: 76) (SEQ ID NO.: 77)  Tg-I CAC CTC CAC CAC CGC AGA GTA TCC TGG CCC ATG CCA TAG AGA (SEQ CAT TGT GAG TTC CCA AGC TCT ID NO.: 79) GTT TTA G (SEQ (SEQ ID NO.: 81) ID NO.: 80) Tg-D GGT TAC GGT GTC AGT TCC AGC TCT CCA GCC TCT AGA AAA CCT GA AAA CAC TTG GTG TTA ATG TGA (SEQ ID NO.: 82) AAG ATG (SEQ ID GCC C (SEQ ID NO.: 83) NO.: 84) *Nucleotides identified in bold and underline are an artificial nucleotide mismatch to the native sequence. ^(¶)This particular pair of forward/reverse primers, named HLA-DRB1*10-2, did not work. †The SE-HR (Shared Epitope-High Risk) qPCR reactions will also include an inhibitor oligonucleotide as follows: ACA TCC TGG AGC AGG CGC GG (SEQ ID NO.: 85).

In a test for MMc in a child, for example, a person of skill in the art may decide to target HLA-DRB1* as an approach to selecting one or more nucleic acid molecule compositions from Table 1. The initial step would be to conduct HLA-genotyping on the child, mother and father. For example, the HLA genotypes may be HLA-DRB1*01/HLA-DRB1*15 for the mother, HLA-DRB1*03/HLA-DRB1*14 for the father, and HLA-DRB1*01/HLA-DRB1*03 for the child. Examination of the HLA-genotyping indicates that the non-shared HLA allele of the mother as compared to the child is HLA-DRB1*03. So, DNA is extracted from the child and interrogated for MMc employing the appropriate HLA-specific Q-PCR assay from among the panel of assays listed in Table 1—in this case, one would use a composition of three nucleic acid molecules that includes SEQ ID NOS.:10, 11, and 12. In addition, an irrelevant HLA-specific Q-PCR assay can be included in testing the child as a negative control.

In certain embodiments, the instant disclosure provides a process for quantitating microchimerism, comprising: (a) amplifying nucleic acid molecules of a target biological sample using one or more specific nucleic acid compositions from Table 1; (b) amplifying nucleic acid molecules of a control biological sample using the one or more specific nucleic acid molecule compositions of step (a); (c) comparing the amount of amplified nucleic acid molecules with a reference to quantitate the specific nucleic acid molecule markers in the samples; wherein the presence of certain specific amplified markers in the target biological sample indicates the presence of microchimerism.

Umbilical cord blood and bone marrow transplants can be used to cure or slow the progression of many cancers originating in the bone marrow (e.g., leukemia, myeloma) or lymphatic system (e.g., lymphoma). In certain embodiments, nucleic acid molecule compositions and methods of the instant disclosure can be used to determine donor MMc to identify one or more donor cord blood or donor bone marrow that will provide the greatest anti-malignancy or anti-relapse potential.

For example, if the HLA-B* is being targeted, then HLA-genotyping would be conducted on the patient, the patient's mother, a first cord blood donor, the first cord blood donor's mother, a second cord blood donor, and the second cord blood donor's mother. The HLA genotypes may be HLA-B*08/HLA-B*15 for the patient, HLA-B*08/HLA-B*44 for the patient's mother, HLA-B*22/HLA-B*15 for the first cord blood donor, HLA-B*22/HLA-B*35 for the first cord blood donor's mother, HLA-B*08/HLA-B*13 for the second cord blood donor, and HLA-B*08/HLA-B*37 for the second cord blood donor's mother. Examination of the HLA-genotyping indicates that the first cord blood donor shares the patient's inherited paternal antigen (IPA), HLA-B*15; therefore, the first cord blood donor would provide the greatest anti-malignancy or anti-relapse potential. By way of illustration and not wishing to be bound by theory, the benefit for the patient to receive the first donor's cord blood would be due to effector T cells of the donor's mother that have previously been exposed to the same HLA molecule as the patient's IPA (i.e., HLA-B*15). In contrast, effector T cells of the mother donor for the second cord blood donor would have reactivity to HLA-B*13, which is not shared with the patient.

If, however, the mother does not have a unique HLA allele (i.e., the mother and child are HLA identical or the child is HLA homozygous) or a set nucleic acid molecules of the instant disclosure are not available, testing can be done on genetic polymorphisms found on other chromosomes. Exemplary nucleic acid molecule compositions from Table 1 include, for example, glutathione S-transferase θ1 (GSTT1), anti-thrombin III long (AT3-L), anti-thrombin III short (AT3-S), thyroglobulin insertion (Tg-I) and thyroglobulin deletion (Tg-D). All nucleic acid molecule compositions have been validated for specificity using Q-PCR assays tested against an extensive panel of DNA derived from fully HLA characterized cell lines from the 13th International HLA Workshop (see www.ihwg.org; see also Marsh et al., Tissue Antigens 75:291, 2010).

In certain medical situations, patients may receive a double cord blood transplant. So in the previous example, although a patient may receive a double cord blood transplant, only one of the donors may provide a benefit. In certain embodiments, the nucleic acid molecule compositions and methods of the instant disclosure can be used to determine which plurality of donor cord blood or donor bone marrow would be best to combine. In a further embodiment, the nucleic acid molecule compositions and methods of the instant disclosure can be used to determine which donor cord blood or donor bone marrow from a pooled transplant would dominantly engraft.

For example, if the HLA-B* is again being targeted, then HLA-genotyping would be conducted on the patient, the patient's mother, a first cord blood donor, the first cord blood donor's mother, a second cord blood donor, and the second cord blood donor's mother. The HLA genotypes may be HLA-B*08/HLA-B*15 for the patient, HLA-B*08/HLA-B*44 for the patient's mother, HLA-B*22/HLA-B*12 for the first cord blood donor, HLA-B*22/HLA-B*35 for the first cord blood donor's mother, HLA-B*18/HLA-B*44 for the second cord blood donor, and HLA-B*18/HLA-B*37 for the second cord blood donor's mother. Examination of the HLA-genotyping indicates that the second cord blood donor shares the patient's non-inherited maternal antigen (NIMA), HLA-B*44; therefore, the second cord blood donor would provide the greatest potential for dominant engraftment in this case, one would use a composition of three nucleic acid molecules that includes SEQ ID NOS.:1, 2, and 3.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DRB1*01, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:4, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:5, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:6.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DRB1*15/16, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:7, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:8, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:9.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DRB1*03, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:10, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:11, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:12.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DRB1*07, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:16, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:17, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:18.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DRB1*08, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:19, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:20, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:21.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DRB1*09, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:22, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:23, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:24.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DRB1*10, wherein (a) the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:25, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:26, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:27.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DRB1*14, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:31, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:32, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:33.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DRB4, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:34, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:35, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:36.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DQA1*01, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:37, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:38, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:39.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DQA1*03, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:40, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:41, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:42.

In further embodiments, the instant disclosure provides a composition comprising comprises nucleic acid molecules specific for HLA-DQA1*05, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:43, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:44, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:45.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DQB1*03, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:49, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:50, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:51.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DQB1*04, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:52, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:53, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:54.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for HLA-DQB1*06, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:55, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:56, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:57.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for SE-HR, wherein (a) the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:58, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:59, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:60, or (b) the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:61, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:62, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:63. In related embodiments, the aforementioned compositions may further comprise an inhibitor nucleic acid molecule comprising a sequence as set forth in SEQ ID NO.:85.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for SE-LR, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:64, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:65, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:66.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for GSTT1, wherein the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:70, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:71, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:72.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for AT3, wherein (a) the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:73, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:74, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:75, or (b) the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:76, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:77, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:78.

In further embodiments, the instant disclosure provides a composition comprising nucleic acid molecules specific for Tg, wherein (a) the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:79, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:80, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:81, or (b) the forward nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:82, the reverse nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:83, and the probe nucleic acid molecule comprises a sequence as set forth in SEQ ID NO.:84.

In any of the aforementioned embodiments, the instant disclosure provides a composition wherein the probe comprises a fluorophore at the 5′-end and a quencher at the 3′-end. In any of the aforementioned embodiments, the instant disclosure provides a composition wherein the fluorophore is FAM and the quencher is TAMRA or BHQ. In any of the aforementioned embodiments, the instant disclosure provides a composition wherein the probe further comprises a duplex stabilizer, such as at least one LNA or MGB.

Although specific embodiments and examples of this disclosure have been described herein for illustrative purposes, various equivalent modifications can be made without departing from the spirit and scope of the invention, as will be recognized by those skilled in the relevant art after reviewing the present disclosure. The various embodiments described can be combined to provide further embodiments. The described devices, systems and methods can omit some elements or acts, can add other elements or acts, or can combine the elements or execute the acts in a different manner or order than that illustrated, to achieve various advantages of the invention. These and other changes can be made to the invention in light of the above detailed description.

In general, in the following claims, the terms used should not be construed to limit the invention to the specific embodiments disclosed in the specification. Accordingly, the invention is not limited by the disclosure, but instead its scope is determined entirely by the following claims

EXAMPLES Example 1 Measuring Allograft Rejection by HLA-Specific Q-PCR of Transplant Recipient Serum

There is no reliable serum marker available to monitor incipient pancreas or islet-cell rejection. Quantification of donor specific genomic DNA in serum was measured as a marker of rejection. A panel of HLA-specific quantitative PCR assays (Q-PCR) was used to test 158 sera from 42 pancreas-kidney transplant recipients. The HLA-specific primers and probes used were directed to DRB1*01 (SEQ ID NOS:4, 5 and 6), DRB1*11 (forward primer CAG ACC ACG TTT CTT GGA GTA CTC TAC, SEQ ID NO.:86; reverse primer CCT TCT GGC TGT TCC AGT ACT CCT, SEQ ID NO.:87, and probe CGC TCC GTC CCA TTG AAG AAA TGA CA, SEQ ID NO.:88), DQA1*01 (SEQ ID NOS:37, 38 and 39), DQA1*03 (SEQ ID NOS:40, 41 and 42), DQB1*02 (SEQ ID NOS:46, 47 and 48), DQB1*03 (SEQ ID NOS:49, 50 and 51), DQB1*06 (SEQ ID NOS:55, 56 and 57), DRB4*01 (SEQ ID NOS:34, 35 and 36), and B*44 (SEQ ID NOS:1, 2 and 3). Temporally related biopsies for 65 sera permitted analysis for correlation of donor DNA concentrations with rejection.

Briefly, a calibration curve for the HLA-specific assay of interest was generated with known quantities of genomic DNA [0, 0.5, 1, 5, 10, 50, 100, and 500 genome-equivalents (gEq)] derived from Epstein-Barr virus-transformed cell lines that were previously HLA typed and known to be homozygous for the allele of interest. A separate β-globin calibration curve was created to quantify the total amount of genomic DNA derived from both host and donor within each specimen. Total genomic DNA was isolated from patient sera (200 to 500 μL) by use of a DNA Mini Kit (Qiagen) with a final elution volume of 50 μL. Specimen HLA Q-PCR reactions contained 10 μL of template DNA (or 5 μL for the β-globin assay to maximize the amount of DNA eluate available for HLA assays), 25 μL of TaqMan® Universal Master Mix (Applied Biosystems), 300 nM each of the forward/reverse primers (MWG Biotech), 100 nM dual labeled probe (MWG Biotech), and DNase/RNase-free water to a final volume of 50 μL. Of note, to prevent PCR competition for the reagents between the more prevalent β-globin PCR product and the less prevalent HLA-specific product, assays were performed in a non-multiplexed format with HLA-specific and β-globin assays contained in separate wells on the same plate. Four wells were measured, on average, for each serum sample for HLA and 1 well for β-globin. PCR reactions were incubated in an ABI Prism 7000 thermocycler for 2 min at 50° C., followed by 45 cycles of 95° C. for 15 s and 60 to 64° C., depending on the HLA assay, for 1 min. HLA (or β-globin) quantities were determined for sample wells by plotting on the calibration curve the point at which a fluorescence threshold for a given assay was exceeded. Results were rejected and assays repeated if either calibration curve correlation coefficient (r2) was <0.99.

HLA quantities were expressed as the total number of cell-free gEq/mL of serum as calculated by the equation: ΣHLA Values/ΣSample Volume×elution volume×1000/serum volume=gEq/mL. The equation accounts for the proportion of the DNA extract that was amplified for each target allele (elution volume/sum of sample volume). In addition, we determined the ratio of donor DNA to host cell-free DNA (as determined by simultaneous β-globin Q-PCR) in the serum to control for nonspecific increases in total soluble DNA. The ratio was subsequently corrected to reflect whether the assayed HLA allele was present in 1 or 2 copies in the donor genome.

Results:

Donor DNA concentrations were higher in sera from recipients who had experienced allograft rejection (n=31) than from those who had not (n=34). Median concentrations, expressed as the genome-equivalent (gEq) number of donor cells per 10⁶ host cells, were 2613 and 59 gEq/10⁶, respectively (P=0.03). Relative concentrations of donor DNA in host serum (gEq/106 host genomes) over time for an exemplary patient probed with DQB1*06 is shown in FIG. 1 .

Conclusion:

Q-PCR for donor-specific genetic polymorphisms is a noninvasive approach to monitor pancreas-kidney, as well as other types of allograft rejection.

Example 2 Measuring Maternal Microchimerism by Hla-Specific Q-PCR

Microchimerism (Mc), originating from bidirectional fetal-maternal cell traffic during pregnancy has been identified in healthy adults and in patients with scleroderma (systemic sclerosis, “SSc”). HLA-specific primers and fluorogenic probes were used in real-time quantitative polymerase chain reaction assays to investigate the frequency and quantitative levels of maternal Mc (“MMc”) in healthy women and women with SSc.

Briefly, HLA-specific primers and probes were used to target non-inherited, non-shared HLA sequences. DNA-based HLA typing was conducted in 67 proband mother pairs and in all children if the proband was parous. Statistical analysis was limited to 50 proband mother pairs (including 32 healthy women and 18 women with SSc) in whom MMc could be distinguished from potential fetal Mc. The probands were either healthy women with no history of autoimmune disease, or women with scleroderma. A total of 253 subjects were studied and included healthy women (n=41) and their mothers (n=41) and children (n=58), and women with SSc (n=26) and their mothers (n=26) and children (n=39). The study population was derived from an overall population of 74 healthy women and 56 women with SSc, among whom 85% had a non-inherited, non-shared maternal HLA-DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, or B allele, with 70% of HLA differences informative using same panel of 8 HLA-specific primers in Q-PCR assays as described in Example 1. Probands who were parous were included in the current study only if all living children were also willing to be studied. This requirement was included because fetal Mc from a prior pregnancy can confound the detection of MMc. Parity was similar in the two groups, and the study subjects were recruited from a similar geographic distribution (state of Washington and surrounding areas). All subjects provided informed consent.

Another potential source of microchimerism is blood transfusion (Lee et al., Blood 93:3127, 1999). The HLA primers used in this study are specific for a particular HLA polymorphism, and chances are extremely low that the blood donor had the same HLA allele as the mother of the proband. Of the 32 controls for whom information on blood transfusion history was available, one had had a blood transfusion prior to the study. Of the 25 scleroderma patients, five had had a blood transfusion (one prior to disease onset, one at the time of onset, and three after onset).

Results.

MMc in peripheral blood mononuclear cells was more frequent among women with SSc (72%) than healthy women (22%) (odds ratio: women with SSc were 9.3 times more likely to have MMc than healthy women, P<0.001) (see Table 2).

TABLE 2 Frequency of MMc in Healthy Women and Women with SSc* No. (%) Odds P vs. Positive Ratio 95% CI Controls Model with all Probands Controls 7/32 (21.9) 9.3 2.5-35 0.001 Cases 13/18 (72.2) Model excluding Probands with transfusions Controls 7/31 (22.6) 6.2 1.6-25 0.01 Cases 9/14 (64.3) *MMc = maternal microchimerism; SSc = systemic sclerosis; 95% CI = 95% confidence internval.

However, levels of MMc, expressed as the genome equivalent of maternal cells per million (gEq/mil), were not significantly different (0-68.6 gEq/mil in SSc patients, 0-54.5 in healthy women) (see Table 3).

TABLE 3 Frequency of MMc in Healthy Women and Women with SSc* gEq/mil No. of host cells, Subjects/ median P vs. Observations (range) Controls Model with all Probands Controls 32/34 0 (0-54.5) 0.056 Cases 18/24 0.71 (0-68.6) Model excluding Probands with transfusions Controls 31/33 0 (0-54.5) 0.18 Cases 14/20 0.71 (0-34.5) *MMc = maternal microchimerism; SSc = systemic sclerosis: gEq/mil = genome equivalent per million.

In additional studies, positivity for MMc was demonstrated in a bone marrow aspirate from an SSc patient in whom peripheral blood had been found to be negative for MMc on four occasions, and tissue from a subsequent autopsy of this patient had MMc levels of 757 and 1,489 gEq/mil in the lung and heart, respectively (see FIG. 2 ).

CONCLUSION

MMc is not uncommon in the peripheral blood of healthy adults, is increased in frequency in patients with SSc, and may be present in bone marrow and disease-affected tissues even if absent in the peripheral blood. 

What is claimed is:
 1. A kit for use in carrying out a method for detecting microchimerism, the kit comprising one or more specific nucleic acid molecule compositions comprising a forward nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 64, a reverse nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 65, and a probe nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 66; wherein the method comprises: (a) amplifying nucleic acid molecules of a target biological sample using the specific nucleic acid molecule compositions; (b) amplifying nucleic acid molecules of a control biological sample using the specific nucleic acid molecule compositions of step (a); (c) comparing the amount of amplified nucleic acid molecules with a reference to quantitate specific nucleic acid molecule markers in the samples; wherein each specific nucleic acid molecule composition amplifies a SE-LR genetic polymorphism; and wherein the presence of certain specific amplified markers in the target biological sample indicates the presence of microchimerism.
 2. A kit for use in detecting incipient allograft rejection from a biological sample of a subject, the kit comprising one or more specific nucleic acid molecule compositions comprising a forward nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 64, a reverse nucleic acid molecule comprising the sequence set forth in SEQ ID NO: 65, and a probe nucleic acid molecule comprising the sequence set forth in SEQ ID NO:
 66. 3. The kit of claim 1, wherein the kit has a detection sensitivity of one chimeric genome in 10⁵ host genomes.
 4. The kit of claim 1, wherein the biological sample is blood or serum.
 5. The kit of claim 1, wherein the amplifying comprises performing Q-PCR.
 6. The kit of claim 2, wherein the biological sample is blood or serum.
 7. The kit of claim 2, wherein the subject is a human.
 8. The kit of claim 2, wherein the subject has a known genotype. 